Pinostrobin, a fingerroot compound, regulates miR-181b-5p and induces acute leukemic cell apoptosis

Pinostrobin (PN) is the most abundant flavonoid found in fingerroot. Although the anti-leukemic properties of PN have been reported, its mechanisms are still unclear. MicroRNAs (miRNAs) are small RNA molecules that function in posttranscriptional silencing and are increasingly being used in cancer therapy. The aims of this study were to investigate the effects of PN on proliferation inhibition and induction of apoptosis, as well as the involvement of miRNAs in PN-mediated apoptosis in acute leukemia. The results showed that PN reduced cell viability and induced apoptosis in acute leukemia cells via both intrinsic and extrinsic pathways. A bioinformatics approach and Protein–Protein Interaction (PPI) network analysis revealed that ataxia-telangiectasia mutated kinase (ATM), one of the p53 activators that responds to DNA damage-induced apoptosis, is a crucial target of PN. Four prediction tools were used to predict ATM-regulated miRNAs; miR-181b-5p was the most likely candidate. The reduction in miR-181b-5 after PN treatment was found to trigger ATM, resulting in cellular apoptosis. Therefore, PN could be developed as a drug for acute leukemia; in addition, miR-181b-5p and ATM may be promising therapeutic targets.


Results
Pinostrobin exhibits cytotoxicity against acute leukemia cell lines. To investigate the cytotoxicity of PN to acute leukemia, the cell viability of NB4 and MOLT-4 leukemia cell lines under PN treatment for 24 and 48 h was observed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. As shown in Fig. 1A, PN significantly reduced the cell viability of both leukemia cell lines in a dose-and timedependent manner. The half-maximal inhibitory concentration (IC50) of PN against NB4 and MOLT-4 at 24 h was 433 ± 84 and 604 ± 157 µM, while the IC50 at 48 h was 132 ± 42 and 142 ± 24 µM, respectively. The viability of peripheral blood mononuclear cells (PBMCs) was unaffected by PN. These findings indicate that PN possesses a cytotoxic effect on NB4 and MOLT-4 leukemia cells without toxicity to healthy PBMCs.
Pinostrobin induces apoptosis in acute leukemia cells. Propidium iodide (PI) staining was used to examine the cell cycle of PN-treated and control cells, followed by flow cytometry detection. The results, presented in Fig. 1B, demonstrated that PN increased the percentage of sub-G1 phase in both NB4 and MOLT-4 cells by 24.63 ± 0.64% and 19.57 ± 0.95%, respectively. These results indicated that PN could induce cell death with fragmented low-molecular-weight DNA in acute leukemia. DNA endonucleolytic cleavage is one of the common features found in apoptosis, a mechanism of cell death that has long been linked to the treatment of cancer. To confirm whether PN exerts any influence on apoptosis in acute leukemia cells, the percentage of apoptotic cells was measured using Annexin V conjugated with fluorescein isothiocyanate (FITC) and PI staining, followed by flow cytometry detection. After 48 h of incubation with the IC50 of PN, the total percentage of apoptotic cells was increased in NB4 and MOLT-4 cells, with values of 35.57 ± 4.23% and 35.27 ± 3.47%, respectively, as shown in Fig. 1C. According to these findings, PN causes acute leukemia cells to undergo apoptosis, which leads to cell death.

Alterations in apoptotic genes in acute leukemia cells after pinostrobin treatment. To investi-
gate the mechanism of PN on the induction of apoptosis in NB4 and MOLT-4 cells, the expression of apoptotic genes, including caspase-3, caspase-8, caspase-9, Fas cell surface death receptor (Fas), and Bcl-2-associated X protein (BAX), was examined after 48 h of treatment with PN at the IC50 using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results are shown in Fig. 1D. PN increased the mRNA expression levels of caspase-3, caspase-8, caspase-9, Fas, and BAX in both cell lines. These results indicated that PNinduced apoptosis in acute leukemia cells involves caspase-dependent mechanisms. Notably, PN activated both intrinsic pathways via BAX and caspase-9 and extrinsic apoptotic pathways via Fas and caspase-8.

Ataxia-Telangiectasia Mutated kinase (ATM) is a pinostrobin-responsive protein.
Using a molecular similarity approach in the ChEMBL database with a threshold of more than 70%, compounds similar to PN were obtained. Due to the similarity principle, 25 target proteins of PN-homologous compounds with bioactivity potency at a concentration less than 150 μM that are expressed in humans were determined as potential targets of PN. These potential targets of PN are displayed in Table 1. Then, these proteins were used for PPI network construction by the STRING database with an intermediate confidence score > 0.4, and the first and second spheres of interaction were 10 and 0, respectively. From network visualization using Cytoscape, the PPI network comprised a total of 110 interactions involving 35 proteins, as presented in Fig. 2A. The hub protein of this network was then examined utilizing the maximal clique centrality (MCC) methods of the CytoHubba application The effect of PN on the mRNA expression of apoptotic genes, including caspase 3, caspase 8, caspase 9, BAX, and Fas, was examined by RT-qPCR. All data was presented as the mean ± SEM of three separate experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 were the statistically significant differences from the control group. www.nature.com/scientificreports/ in Cytoscape. Figure 2B displays the top 10 proteins with the highest MCC score, which included ATM, CHEK2, PCNA, TP53, CHEK1, TP53BP1, MCM6, CDT1, APEX1, and NBN. Based on the gene ontology (GO) analysis  via the STRING database, 14 proteins, including DUSP6, RPS6KA1, PIM1, PIN1, MAPK1, APEX1, TP53, NBN,  CHEK1, CHEK2, ATM, VDR, CYP1B1, and TARDBP, function in the apoptosis process. As shown in Fig. 2C, the hub proteins of this network that most often interacted with other proteins in the network and were involved in the apoptotic process included ATM, TP53, CHEK2, CHEK1, APEX1, and NBN. Among these 6 proteins, ATM exhibited the highest MCC score. This result indicated that ATM might be an important target protein of PN in inducing apoptosis in acute leukemia cells. The KEGG pathway database was then used to identify the ATM downstream signaling pathway. The findings showed that ATM contributed to the apoptotic process by activating the p53 signaling pathway.

ATM and p53 are involved in apoptosis induction by pinostrobin in acute leukemia cells.
The effect of PN on ATM and p53 expression was investigated to verify the network analysis findings. Leukemia cells were treated with PN at the IC50 for 48 h, followed by detection of ATM and p53 expression via RT-qPCR and western blot analysis. The results shown in Fig. 2D revealed that PN increased the mRNA expression levels of ATM and p53 in both leukemia cell lines. These results were consistent with the western blot results shown in Fig. 2E, which similarly demonstrated the elevation of ATM after 48 h of PN treatment. These findings supported the hypothesis that PN can promote apoptosis by upregulating ATM, which further activates the p53 signaling pathway.
miR-181b-5p is predicted to be an ATM-specific miRNA. The miRNAs that regulate ATM were predicted by four different prediction tools. There were 288, 154, 107, and 1900 miRNAs obtained from DIANA, miRDB, TargetScanHuman, and RNA22, respectively. In accordance with Fig. 3A, 73 miRNAs, consisting of 12 miRNAs retrieved from all prediction tools and 51 miRNAs predicted from three prediction tools, were obtained. Next, the miRNAs were manually selected based on the selection criteria described in the materials and methods. Overall, 22 possible ATM-regulated miRNAs met all the criteria. The target accessibility of each possible ATM-regulated miRNA was evaluated using Sfold. From a probability histogram in Fig. 3B, positions of nucleotides with a probability greater than 0.5 were defined as accessible regions for miRNA binding. Fourteen candidate miRNAs for ATM suppression, listed in Table 2, could hybridize to the accessible regions of ATM mRNA. These miRNAs were then reviewed for their supportive evidence in inhibiting ATM. Strong supporting evidence provided by RT-qPCR, western blot, or luciferase test results confirmed the regulation of ATM www.nature.com/scientificreports/ The association between the exposure to PN and the mRNA expression levels of ATM and p53 was examined using RT-qPCR. (E) The protein expression level of ATM was determined by western blot analysis. Uncropped blots were included in Supplement 3. Data was expressed as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 were the statistically significant differences from the control group. www.nature.com/scientificreports/ The expression level of miR-181b-5p after treatment with the IC50 concentration of PN was examined using RT-qPCR. Data was expressed as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 were the statistically significant difference from the control group. www.nature.com/scientificreports/ by miR-26a-5p, miR-26b-5p, miR-27a-5p, miR-181a-5p, and miR-181b-5p [23][24][25][26][27][28] . Among the 5 miRNAs, miR-181b-5p was chosen because it had the lowest ∆G hybrid value and the highest LogitProb value.

miR-181b-5p is downregulated in pinostrobin-treated leukemia cells. To verify the result from
the prediction of ATM-regulated miRNAs, the expression level of miR-181b-5p was investigated via RT-qPCR. NB4 and MOLT-4 cells were treated with the IC50 of PN for 6, 12, 24, and 48 h. The results presented in Fig. 3C demonstrate that PN significantly suppressed the expression level of miR-181b-5p at 48 h in NB4 and MOLT-4 cells. This result indicated that miR-181b-5p is involved in the mechanism of PN-induced apoptosis in acute leukemia cells.

miR-181b-5p is involved in PN-induced apoptosis in acute leukemia cells.
The miR-181b-5p mimic was transiently transfected into leukemia cells alone and in combination with PN at the IC50 to determine whether miR-181b-5p expression levels have any influence on the expression of ATM and p53 and the activation of apoptosis. Leukemia cells transfected with the microRNA mimic DS control served as the control group. To determine the efficiency of the transfection processes, the expression of miR-181b-5p was investigated using RT-qPCR. The results in Fig. 4A demonstrate that the expression of miR-181b-5p was increased in cells transfected with miR-181b-5p mimics alone compared to the control group. However, the expression of miR-181b-5p in cells that were transfected with miR-181b-5p mimic followed by PN treatment was decreased.
The results showed that in miR-181b-5p-overexpressing cells, PN was still effective in inhibiting miR-181b-5p expression. Then, to examine the regulatory effect of miR-181b-5p on ATM and p53, transfected cells under different conditions were harvested to measure ATM and p53 expression levels using RT-qPCR. Figure 4B shows that ATM and p53 were repressed in miR-181b-5p-mimic-transfected cells. This outcome supported the role of miR-181b-5p in ATM inhibition. The expression of ATM and p53 increased after miR-181b-5p mimic transfection with PN therapy. These results implied that PN might silence miR-181b-5p to activate the ATM and p53 signaling pathways. The expression of ATM at the mRNA level also correlated with its expression at the protein level, which was investigated by western blot analysis (Fig. 4C). The percentage of apoptosis was determined using Annexin V-FITC and PI staining to study the impact of miR-181b-5p on apoptosis induction. The percentage of apoptosis in miR-181b-5p-transfected cells was not different from that in the control group, as shown in Fig. 4D. However, the percentage of apoptosis increased in cells that were transfected with miR-181b-5p mimic followed by PN treatment. These results suggested that PN induces apoptosis in leukemia cells by regulating miR-181b-5p expression.

Discussion
Pinostrobin is a major phytochemical in the group of flavonoids found in fingerroot (Boesenbergia rotunda) 8 . In this study, we explored the anticancer properties of PN in NB4 and MOLT-4 acute leukemia cells. PN reduced the viability of both leukemia cell lines via apoptosis induction, resulting in an increase in the proportion of cells in the sub-G1 phase of the cell cycle and the percentage of apoptotic cells. These results correlated with the outcomes of previous studies that investigated the anticancer activities of PN in acute leukemia (Jurkat and HL60) 14 , chronic leukemia (K562) cells 12 , and other types of cancer, including cervical cancer 13 , breast cancer and glioblastoma 12 . Although there is evidence of the anti-leukemic activity of PN, the underlying mechanism of its role in apoptosis induction is still unclear. Therefore, the effect of PN on apoptotic gene expression was investigated. Caspase-8 and caspase-10 are engaged in the extrinsic apoptotic pathway, while caspase-9 triggers the intrinsic apoptotic pathway. The activation of these initiator caspases further activates caspase-3, leading to apoptosis 29 . Our results revealed that caspase-3, caspase-8, and caspase-9 were upregulated in PN-treated cells www.nature.com/scientificreports/ Data was expressed as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 were the statistically significant difference from the control group. # P < 0.05, ## P < 0.01 and ### P < 0.001 were the statistically significant difference from the miR-181b-5p mimic transfected group. www.nature.com/scientificreports/ compared to control cells. Moreover, PN was shown to increase the expression of BAX and Fas, a proapoptotic protein that plays a role in the intrinsic pathway of apoptosis and a death receptor that is important for the extrinsic apoptotic pathway, respectively. These findings indicated that PN induces apoptosis in acute leukemia cells via the intrinsic and extrinsic apoptotic pathways by activating the caspase family (caspase-3, 8, and 9), pro-apoptotic protein (BAX), and death receptor (Fas). A toxicity study of PN was performed in Wistar rats, demonstrating that PN is nontoxic at doses between 1 and 100 mg/kg 30 . According to previous studies, our research also revealed that PN had a lesser effect on PBMCs from healthy subjects. To further understand the underlying mechanism of PN-mediated apoptosis, the target protein of PN was identified using the ligand-based approach and the PPI network. We obtained 25 potential target proteins of PN from the ChEMBL database via the similarity principle. These proteins, together with 10 others from the STRING database, were used to construct the PPI network. Among these 35 proteins, 14 function in the apoptosis process. The MCC method can be used to identify essential proteins from complex interactomes 31 . Because ATM had the highest MCC score, it might be the key target protein of PN in apoptosis induction. According to the findings of the KEGG pathway study as well as other research 32 , ATM has been demonstrated to mediate the activation of the apoptotic pathway in response to DNA damage. Many previous studies reported that ATM phosphorylates p53 to stabilize p53, and then the phosphorylated p53 will further activate the expression of a panel of apoptotic genes and facilitate both intrinsic and extrinsic apoptotic pathways. Recently, in response to DNA damage, ATM can promote the expression of p53 mRNA and the synthesis of p53 protein by phosphorylating MDM2 at Ser395. This phosphorylation brings MDM2 to stabilize the p53 mRNA and recruits ATM to the p53 polysome to phosphorylate the nascent p53 protein and prevents newly synthesized p53 from MDM2-mediated degradation 33,34 . To verify the prediction from the PPI network, the expression of ATM and p53 was determined using RT-qPCR and western blot analysis. NB4 and MOLT-4 displayed overexpression of ATM and p53 after PN treatment. These results suggest that PN can promote intrinsic and extrinsic apoptotic pathways by upregulating ATM, which further activates the p53 signaling pathway. Several studies have reported that the p53 signaling pathway and ATM involved in apoptosis can be induced by natural compounds. For example, the fungal metabolite galiellalactone induces cell cycle arrest and apoptosis in prostate cancer cells via ATM phosphorylation 35 . Bavachinin activates ATM to induce apoptosis in small cell lung cancer cells 36 . In addition, curcumin, an active ingredient of turmeric, shows antitumor activity by targeting the ATM/Chk2/p53 signaling pathway 37 .
MicroRNAs have been mentioned as epigenetic factors controlling the posttranscriptional regulation of gene expression. They control a wide range of cellular functions, including growth, proliferation, and degeneration. Recently, the involvement of miRNAs in phytochemical-mediated apoptosis has received much attention 16 . We applied bioinformatics methods from Seenprachawong et al. 38 to predict miRNAs related to PN-induced apoptosis in acute leukemia. One miRNA discovered using bioinformatics was miR-181b-5p, a member of the miR-181 family, which is highly evolutionarily conserved in almost all vertebrates. This miRNA family consists of four members, including miR-181a, miR-181b, miR-181c, and miR-181d 39 . Many studies have demonstrated that the miR-181 family regulates the differentiation of granulocytic and macrophage-like cells by targeting PRKCD, CTDSPL, and CAMKK. The miR-181 family was also found to be a marker of common myeloid and erythroid progenitor commitment 40 . There is evidence that miR-181 is overexpressed in several malignancies, including acute myeloid leukemia, colorectal cancer, and breast cancer [41][42][43] . Our study showed that miR-181b-5p acts as an oncogene by regulating ATM and p53. By transiently transfecting the miR-181b-5p mimic into acute leukemia cells, the expression of ATM and p53 was repressed. To the contrary, the reduction of miR-181b-5p by PN increased the expression of ATM. These results were consistent with previous studies from Yujun Wang and Andrea BISSO 23,44 . They showed that the ectopic overexpression and inhibition of miR-181a/b, respectively, were related to the decreased and enhanced expression of ATM at both the mRNA and protein levels. Also, they used a luciferase reporter assay to confirm that the 3′ UTR of ATM was a direct target of miR-181a/b. Moreover, previous studies revealed that overexpression of the miR-181 family showed a strong correlation with overall survival and aggressiveness of cancers, including leukemia 23,41 .
The present study demonstrated the anti-leukemic effects of PN on NB4 and MOLT-4 cell lines since PN can suppress miR-181b-5p, increase the expression of ATM and p53, and trigger apoptosis in acute leukemia cells, including those with ectopically expressed miR-181b-5p. These results highlighted the ability of PN to be used therapeutically for leukemia in the future. Additionally, these findings may be beneficial for improving our understanding of the role of miR-181b-5p in human leukemia.

Methods
Leukemia cell culture. The human acute promyelocytic leukemia cell line (NB4) and the human acute lymphocytic leukemia cell line (MOLT-4) were purchased from Cell Lines Service (Eppelheim, Germany). Leukemia cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) FBS and 1% (v/v) penicillinstreptomycin (Gibco Life Technologies, Waltham, MA, USA). Cells were maintained in a humidified incubator at 37 °C with 5% CO 2 . The medium was changed every 2-3 days.

PPI network construction and Identification of PN-responsive proteins.
Compounds sharing more than 70% of a chemical structure similar to PN were obtained from the ChEMBL database (https:// www. ebi. ac. uk/ chembl/) 45 . Based on the similarity principle, structurally similar compounds may share common targets. Therefore, targets of PN-homologous compounds may likewise be potential targets of PN. Then, these discovered target proteins were chosen for PPI network construction to observe how these proteins interact and identify the most probable target of PN in the induction of apoptosis. The chosen proteins must be expressed in Homo sapiens and are target proteins for substances with bioactivity potency at concentrations less than 150 μM. The PPI network was constructed by the STRING database (http:// string-db. org) 46 . Protein and gene interaction networks in biological organisms are frequently scale-free networks that obey the power law of P(k) ~ k −γ47 . In order to obtain a scale-free network which is an optimal network, the network was set up with an intermediate confidence score of more than 0.4 and the first and second spheres of interaction were varied from 10 to 100 nodes. All networks were exported to Cytoscape software to analyze network topology 48,49 . The γ and R-square in correlation with the power law of all networks were listed as Supplement 1. The highest R-squared was found in the network with 10 first sphere nodes and no second sphere nodes. As a result, this network was chosen for further investigation. A gene ontology (GO) analysis of this network was performed by the STRING database to study the underlying mechanism of PN in the induction of apoptosis. The PN-responsive protein was identified using the Maximal Clique Centrality (MCC) calculation method of the CytoHubba application in Cytoscape 31 54 , and miRDB (http:// mirdb. org) 55 , in an updated version, were used for predicting miRNAs that target ATM. Only miRNAs that (1) were predicted by more than or equal to three prediction tools; (2) had a p-value less than 0.05 if obtained from RNA22; (3) had a prediction score greater than 80 if obtained from miRDB; (4) had a miTG score greater than or equal to 0.7 if obtained from DIANA-tools; and (5) had a context + score less than or equal to-0.1 or P CT greater than or equal to 0.5 if obtained from TargetScanHuman were selected. The miRNAs were then examined using Sfold (http:// sfold. wadsw orth. org/ starm ir. html) to determine their target accessibility 56 . A sequence of the 3′UTR of the ATM mRNA was used as an input to obtain a probability histogram. Since a high probability will increase the chance of successful miRNA binding, the particular positions of nucleotides with a probability greater than 0.5 were defined as an accessible region for miRNA 38 . miRNAs that could hybridize with the accessible region were categorized as candidate miRNAs for inhibition of the ATM gene. The candidate miRNAs were reviewed for their supportive evidence in targeting ATM before being selected for further experiments.
miR-181b-5p mimics transfection. 1  www.nature.com/scientificreports/ 1 μM using serum-free Opti-MEM™ medium as a diluent. Then, the diluted miR-181b-5p mimic was mixed with the diluted lipofectamine 3000™ at a 1:1 ratio (v/v Determination of gene expression and miR-181b-5p expression by reverse transcription-quantitative PCR (RT-qPCR). Cells were harvested by centrifugation at 3500 rpm for 5 min. Total RNA was extracted using the GENEzol™ reagent (New England Biolab, Inc., Ipswich, MA, USA). For miRNA, RNA was extracted using Direct-zol RNA Miniprep Kits (Zymo Research, Irvine, CA, USA) according to the manufacturer's instructions. RNA concentration was measured by the Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). The Thermo Scientific RevertAid first-strand cDNA synthesis kit (Thermo Scientific, Waltham, MA, USA) was used to synthesize cDNA for investigating mRNA expression while the miRNA 1ststrand cDNA Synthesis Kit (Agilent Technologies, Santa Clara, CA, USA) was used to synthesize cDNA for miRNA expression. The primers were designed from literature reviews and their quality was determined by the Primer-BLAST 57 and the BLASTN database 58 . The primer sequences used in this study are showed in Supplement 2. Then, qPCR was performed using the designed primers, Luna ® Universal qPCR Master Mix (New England Biolab, Inc., Ipswich, MA, USA) and CFX Connect Real-Time PCR Detection System (Bio-Rad, Inc., Hercules, CA, USA). The relative mRNA and miRNA expression of target genes were analyzed by Bio-ad CFX Manager software (Bio-Rad, Inc., Hercules, CA, USA) using the 2 −∆∆CT method 59 . GAPDH was used to normalize mRNA expression and U6 was used to normalize miRNA expression.

Western blot analysis.
After being harvested, the cells underwent two 1X PBS washes. The whole cell lysate was prepared by incubating the cells for 15 min in 0.1 ml of cold RIPA lysis buffer containing 1% protease inhibitor, followed by centrifugation at 14,000×g for 10 min at 4 °C. The Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, Inc., Hercules, CA, USA) was used to measure the protein concentration in accordance with the manufacturer's instructions. Then, equal amounts of proteins (10 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitro cellulose membranes. Following that, membranes were incubated at 4 °C for an overnight period with monoclonal mouse anti-ATM and anti-β-actin. The membranes were then immunoblotted with HRP-conjugated horse anti-mouse IgG antibody at 37 °C for 90 min. The signal was developed with an enhanced chemiluminescence (ECL) substrate and detected chemiluminescence by the ChemiDoc™ MP Imaging System. Band density was quantitated using the Image Lab™ software.
Statistical analysis. Three duplicates of each experiment were carried out. The findings were displayed as mean ± standard error of mean (SEM). Every statistical analysis was done using SPSS (SPSS Inc., Chicago, IL, USA). The student t-test was used to compare the results between the two groups, and one-way ANOVA was used to compare the results between more than two groups. Statistical significance was defined as a p-value of less than 0.05.

Data availability
The datasets generated and/or analyzed during the current study are available in the GenBank repository, [Accession number: NM_000051, https:// www. ncbi. nlm. nih. gov/ nucco re/ NM_ 000051. 4? report= genba nk]. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact Dalina Tanyong (dalina.itc@mahidol.ac.th).